Evaluation of anti-infective potential of a tribal folklore Odina wodier Roxb against some selected microbes and herpes simplex virus associated with skin infection.

AIM: To evaluate the in vitro antimicrobial activity of aqueous and methanol extracts of Odina wodier bark (OWB), a folk medicine, against representative bacteria, fungi and herpes simplex virus (HSV) associated with skin infections. METHODS AND RESULTS: The OWB extract(s) was found to inhibit the isolates of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli at an MIC of 256-5000 µg ml(-1) and Candida albicans at and above 4000 µg ml(-1) by agar and broth dilution assays. The growth curve of Staph. aureus revealed the highest activity within 2-6 h of methanol extract (ME) exposure. Interestingly, the MTT and plaque reduction assay showed that the extracts can inhibit HSV-1 and HSV-2 at EC50 of 22·4 and 28·8 µg ml(-1) , with Selectivity index of 11·7-15. While the time kinetic and binding assays demonstrated that the ME at 50 µg ml(-1) prevents viral attachment into Vero cells. Phytochemical and HPLC analysis of ME revealed the presence of flavonoids, phytosterols, saponins and tannins including the pseudotannin chlorogenic acid. CONCLUSION: The traditional use of OWB for the management of skin infections has scientific basis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the antimicrobial potential of OWB on selected isolates of bacteria, fungi and HSV, associated with skin infections.

Anti-apoptotic, anti-inflammatory, and immunomodulatory activities of 3,3'-diselenodipropionic acid in mice exposed to whole body γ-radiation.

The present study was designed to evaluate the possible protective effects of 3,3'-diselenodipropionic acid (DSePA), a potent radioprotector, against oxidative organ damage induced by whole body γ-irradiation and explore its mechanistic effects. The mice were subjected to whole body γ-irradiation at 5 Gy for the detection of oxidative stress, apoptosis, and proliferation in the intestinal (jejunum) tissue and at 7 Gy for the examination of intestinal inflammation and immune imbalance. Groups of mice received intraperitoneal injections of DSePA (2 mg/kg/day) or vehicle (phosphate-buffered saline) for 5 consecutive days prior to irradiation. The whole body γ-irradiation of mice led to the induction of oxidative stress and apoptosis in the intestinal tissue, and pretreatment with DSePA significantly reduced both these parameters. It was also found to abrogate the radiation-induced intestinal inflammatory response and augment the proliferation of intestinal cells. Additionally, irradiation-induced polarization of Th1/Th2 immune balance toward the Th2-dominant direction and pretreatment with DSePA ameliorated this shift, which may be beneficial for the recovery from radiation injury. In conclusion, pretreatment with DSePA prevented radiation-induced oxidative damage in small intestine and the underlying mechanisms responsible for this could be attributed to inhibition of oxidative stress, apoptosis, and inflammation.

Protective effects of selenocystine against γ-radiation-induced genotoxicity in Swiss albino mice.

Selenocystine (CysSeSeCys), a diselenide aminoacid exhibiting glutathione peroxidase-like activity and selective antitumor effects, was examined for in vivo antigenotoxic and antioxidant activity in Swiss albino mice after exposure to a sublethal dose (5 Gy) of γ-radiation. For this, CysSeSeCys was administered intraperitoneally (i.p.) to mice at a dosage of 0.5 mg/kg body weight for 5 consecutive days prior to whole-body γ-irradiation. When examined in the hepatic tissue, CysSeSeCys administration reduced the DNA damage at 30 min after radiation exposure by increasing the rate of DNA repair. Since antigenotoxic agents could alter the expression of genes involved in cell cycle arrest and DNA repair, the transcriptional changes in p53, p21 and GADD45α were monitored in the hepatic tissue by real-time PCR. The results show that CysSeSeCys alone causes moderate induction of these three genes. However, CysSeSeCys pretreatment resulted in a suppression of radiation-induced enhancement of p21 and GADD45α expression, but did not affect p53 expression. Further analysis of radiation-induced oxidative stress markers in the same tissue indicated that CysSeSeCys significantly inhibits lipid peroxidation and prevents the depletion of antioxidant enzymes and glutathione (GSH) levels. Additionally, it also prevents radiation-induced DNA damage in other radiation sensitive cellular systems like peripheral leukocytes and bone marrow, which was evident by a decrease in comet parameters and micronucleated polychromatic erythrocytes (mn-PCEs) frequency, respectively. Based on these observations, it is concluded that CysSeSeCys exhibits antigenotoxic effects, reduces radiation-induced oxidative stress, and is a promising candidate for future exploration as a radioprotector.

Aqueous synthesis of ZnTe/dendrimer nanocomposites and their antimicrobial activity: implications in therapeutics.

The present strategy proposes a simple and single step aqueous route for synthesizing stable, fluorescent ZnTe/dendrimer nanocomposites with varying dendrimer terminal groups. In these hybrid materials, the fluorescence of the semiconductor combines with the biomimetic properties of the dendrimer making them suitable for various biomedical applications. The ZnTe nanocomposites thus obtained demonstrate bactericidal activity against enteropathogenic bacteria without having toxic effects on the human erythrocytes. The average size of the ZnTe nanoparticles within the dendrimer matrix was in the range of 2.9-6.0 nm, and they have a good degree of crystallinity with a hexagonal crystal phase. The antibacterial activities of the ZnTe/dendrimer nanocomposites (ZnTe DNCs) as well other semiconductor nanocomposites were evaluated against enteropathogenic bacteria including multi-drug resistant Vibrio cholerae serogroup O1 and enterotoxigenic Escherichia coli (ETEC). ZnTe DNCs had significant antibacterial activity against strains of V. cholerae and ETEC with minimum inhibitory concentrations ranging from 64 to 512 µg ml(-1) and minimum bactericidal concentrations ranging from 128 to 1000 µg ml(-1). Thus, the observed results suggest that these water-soluble active nanocomposites have potential for the treatment of enteric diseases like diarrhoea and cholera.

Effect of functional groups on antioxidant properties of substituted selenoethers.

Selenoethers attached to functional groups through propyl chain viz., bis(3-carboxypropyl)selenide (SeBA), bis(3-hydroxypropyl)selenide (SePOH) and bis(3-aminopropyl)selenide dihydrochloride (SePAm), have been examined for their ability to inhibit peroxyl radical mediated DNA damage, peroxyl radical scavenging ability and glutathione peroxidase (GPx) like activity. The DNA damage was monitored by gel electrophoresis, bimolecular rate constants for scavenging of model peroxyl radical were determined by pulse radiolysis and the GPx activity was followed by their ability to reduce hydrogen peroxide in the presence of glutathione utilizing NADPH decay and HPLC analysis. Among these compounds, SeBA showed maximum DNA protecting activity and it was also the most efficient in scavenging peroxyl radicals with the highest GPx mimicking activity. Quantum chemical calculations confirmed that SeBA with the highest energy level of HOMO (highest occupied molecular orbital) is the easiest to undergo oxidation and therefore exhibits better radical scavenging, GPx mimicking and DNA protecting activity than SePOH or SePAm.

In vivo radioprotection studies of 3,3'-diselenodipropionic acid, a selenocystine derivative.

3,3'-Diselenodipropionic acid (DSePA), a diselenide and a derivative of selenocystine, was evaluated for in vivo radioprotective effects in Swiss albino mice, at an intraperitoneal dose of 2 mg/kg body wt, for 5 days before whole-body exposure to gamma-radiation. The radioprotective efficacy was evaluated by assessing protection of the hepatic tissue, the spleen, and the gastrointestinal (GI) tract and survival against sub- and supralethal doses of gamma-radiation. DSePA inhibited radiation-induced hepatic lipid peroxidation, protein carbonylation, loss of hepatic function, and damage to the hepatic architecture. DSePA also attenuated the depletion of endogenous antioxidants such as glutathione, glutathione peroxidase, superoxide dismutase, and catalase in the livers of irradiated mice. DSePA also restored the radiation-induced reduction in villus height, crypt cell numbers, and spleen cellularity, indicating protective effects on the GI tract and the hematopoietic system. The results from single-cell gel electrophoresis of the peripheral blood leukocytes showed that DSePA can attenuate radiation-induced DNA damage. The mRNA expression analysis of genes revealed that DSePA augmented GADD45alpha and inhibited p21 in both spleen and liver tissues. DSePA also inhibited radiation-induced apoptosis in the spleen and reversed radiation-induced alterations in the expression of the proapoptotic BAX and the antiapoptotic Bcl-2 genes. In line with these observations, DSePA improved the 30-day survival of irradiated mice by 35.3%. In conclusion, these findings clearly confirm that DSePA exhibits protective effects against whole-body gamma-radiation and the probable mechanisms of action involve the maintenance of antioxidant enzymes, prophylactic action through the attenuation of the DNA damage, and inhibition of apoptosis.

Effect of Phyllanthus niruri Linn. treatment on liver, kidney and testes in CCl4 induced hepatotoxic rats.

Phyllanthus niruri extract is extensively used in treating liver ailments. Effects of aqueous extract of P. niruri on liver, kidney and testes of CCl4 induced hepatotoxic rats were studied. High levels of malondialdehyde (MDA) were observed in the CCl4 test group with significant reduction of MDA levels in all groups on P. niruri extract administration. Highest levels of glutathione (GSH) were found in P. niruri group. Activities of alanine transaminase, aspartate transaminase and alkaline phosphatase enzymes were significantly reduced in the curative group (P. niruri treatment after CCl4 injection). Histopathology of liver showed lesser degree of inflammation in all P. niruri treated groups while the renal and seminiferous tubules showed eosinophilic protein casts with signs of tubular damage and degeneration. Testes also showed decreased amount of mature spermatozoa. The results suggest that P. niruri has anti-oxidant and hepato-protective activity with associated deleterious effects on kidney and testes.

Genomic diversity among Vibrio cholerae O139 strains isolated in Bangladesh and India between 1992 and 1998.

In order to assess the extent of genomic diversity among Vibrio cholerae O139 strains, restriction fragment length polymorphisms in two genetic loci, rrn and ctx, were studied. Analysis of 144 strains isolated from different regions of Bangladesh and India between 1992 and 1998 revealed the presence of at least six distinct ribotypes (B-I through B-VI) of which three were new ribotypes, and one of these was represented by a nontoxigenic O139 strain. Strains of ribotypes B-I through B-V shared 11 different CTX genotypes (A through K). Antimicrobial resistance patterns of the strains varied independently of their ribotypes and CTX genotypes. Results of this study suggest that V. cholerae O139 is undergoing rapid genetic changes leading to the origination of new variants, and temporal changes in antimicrobial resistance patterns may be contributing to the selection of different variants.

Clonal diversity among recently emerged strains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread.

The genomes of the O3:K6 strains of Vibrio parahaemolyticus which abruptly emerged in Calcutta, India, in February 1996 and which demonstrated an unusual potential to spread and an enhanced propensity to cause infections were examined by different molecular techniques to determine clonality. No restriction fragment length polymorphism (RFLP) in the gene encoding the thermostable direct hemolysin was observed among the O3:K6 isolates of V. parahaemolyticus. Clonal diversity among the O3:K6 strains became evident by examining the RFLPs of the rrn operons and by the use of pulsed-field gel electrophoresis. Five ribotypes were distinguished among the O3:K6 strains examined, with ribotype R4 constituting the major type. Strains of O3:K6 isolated between June and August 1996 showed different pulsotypes compared to the pulsotypes of strains isolated before and after this period, indicating genetic reassortment among these strains, but those isolated between August 1996 and March 1998 showed identical or nearly similar pulsotypes. It is clear that there is a certain degree of genomic reassortment among the O3:K6 clones but that these strains are predominantly one clone.

Topological shape and size of peptides: identification of potential allele specific helper T cell antigenic sites.

A database of primary sequences of 28 immunogenic peptides, known to elicit T cell response, derived from five different haplotypes was compiled to identify allele specific helper T cell antigenic sites using a rule based graph-theoretical method. The prediction was based on the identification of allele specific patterns in the form of "topological shape and size" present in the peptides. Indices computed from weighted connected graph models of amino acid side chains and peptides were used in this purpose. The system was trained by 10 Ad and 10 non-Ad restricted peptide sequences, assigned actives and inactives, respectively, chosen randomly from the database, and four Ad and four non-Ad restricted sequences were kept as test peptides. This allowed the system to learn about "topological shape and size" specific for Ad restricted peptides from the differences, if any, they had with the inactive peptides in that respect. The system made 100% correct prediction for the training set peptides and misclassified only one inactive peptide of the test set. The system also identified crucial residues for lambda repressor 12-24 and insulin A-chains. This identification also shows that activity related/crucial residues could be located at varying distances from the peptide terminals. To our knowledge, the method is unique of its kind in the literature and may find application in the rational design of synthetic vaccines and other peptides of immunological importance.

Rapid spread of the new clone of Vibrio cholerae O1 biotype El Tor in cholera endemic areas in India.

Using molecular techniques, we investigated whether the clone of Vibrio cholerae O1 biotype El Tor which appeared in Calcutta, India, in 1994 has spread to other cholera endemic areas in the country. The ribotype of 31 of the 33 strains isolated from different parts of India during 1996 and 1997 was identical to the ribotype displayed by the new clone of V. cholerae O1 which emerged in Calcutta in 1994. Likewise, 12 of the 15 strains examined by pulsed-field gel electrophoresis (PFGE) showed identical profile to that exhibited by the new clone of O1. The restriction fragment length polymorphism (RFLP) of CTX genetic element of these strains also matched with the new clone of O1 which emerged after the outbreak of V. cholerae 0139 in Calcutta. However, two strains (AH042 and AH046) isolated from an outbreak in Ahmedabad (western India) showed different CTX RFLP but had the same ribotype and PFGE profile as the new clone, whereas one strain from Goa (G2) showed distinct ribotype and PFGE profile and the CTX RFLP was identical to the O1 strains which prevailed before the genesis of 0139 in Calcutta. The drug resistance pattern of most of the O1 strains examined in this study, except strain G2, was similar to that of the new clone of V. cholerae O1. None of the strains in this study carried plasmids. Molecular studies clearly show that the new expanded drug resistant clone of V. cholerae O1 has spread to all cholera endemic areas in India and also provide evidence for the evolution of new clones of the O1 serogroup.

Molecular epidemiology of reemergent Vibrio cholerae O139 Bengal in India.

We report the prevalence of the O139 serogroup in Calcutta, India, after its reemergence in August 1996 and the spread of the reemerged clone to other parts of the country by using previously established molecular markers. Phenotypically, the reemerged Vibrio cholerae O139 displayed a difference compared to those that appeared in late 1992 and 1993 in that the current O139 strains are sensitive to co-trimoxazole. Ribotyping with the enzyme BglI produced two rRNA restriction patterns in the O139 strains isolated after August 1996, and these patterns were identical to those exhibited by strains of O139 isolated in 1992. Three clones of V. cholerae O139 are currently prevailing in the country, with strains exhibiting three bands after HindIII digestion and hybridization with a ctxA probe being dominant. The reemergence of V. cholerae O139 in Calcutta after a 32-month quiescent period reestablishes the O139 serogroup as an entity which is likely to play a crucial role in the temporal antigenic variations among the serogroups of V. cholerae causing cholera.

Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal.

Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K. Waldor and J.J. Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity. Both probes did not hybridize with any strain of V. cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae. Among the 126 strains of V. cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains. Both probes were found to be highly specific and sensitive and can be used for the specific identification of V. cholerae O139.

Distribution of genes encoding cholera toxin, zonula occludens toxin, accessory cholera toxin, and El Tor hemolysin in Vibrio cholerae of diverse origins.

A large collection of 1154 strains of Vibrio cholerae of diverse origins including serogroups 01 and 0139 and those belonging to the non-01 and non-0139 (non-01:non-0139) serogroups were examined with a battery of DNA probes specific for cholera toxin (CT), zonula occludens toxin (ZOT), accessory cholera toxin (ACE) and El Tor hemolysin (HLY) to determine the distribution of genes among wild strains and to understand the importance of these factors in the pathogenesis of the disease cholera. Among the 01 clinical isolates, the majority of the strains had an intact core region (ctx, zot, ace) and also possessed the hlyA gene. Although rare, strains of 01 with natural deletions of the ctx, zot and/or ace genes were also detected. The absence of the virulence genes comprising the core region and the presence of the hlyA gene dominated the 01 environment, food isolates and the clinical and environmental non-01: non-0139 strains of V. cholerae. All the 0139 strains examined in this study possessed genes located in the core region and the hlyA gene. Among all the virulence-associated genes examined, the hlyA gene was the most conserved genetic element in V. cholerae independent of biotypes and serogroups.

Evidence for the presence of a receptor for the cytolethal distending toxin (CLDT) of Campylobacter jejuni on CHO and HeLa cell membranes and development of a receptor-based enzyme-linked immunosorbent assay for detection of CLDT.

Using ligand blotting, it was found that partially purified cytolethal distending toxin prepared from an enterotoxigenic strain of Campylobacter jejuni, bound to two peptides of molecular masses of approximately 59 kDa and 45 kDa and to a single peptide of 59 kDa in protein blots prepared from HeLa and CHO cell membranes, respectively. In contrast, labile toxin of Escherichia coli and cholera toxin bound to a single peptide of the same molecular mass (15 kDa) on protein blots prepared from both CHO and HeLa cell crude membranes resolved by gel electrophoresis. This banding pattern was identical using SDS-solubilized membrane, with or without heat treatment, but no band was obtained when reduced (treatment with 2-mercaptoethanol) samples were used for the gel electrophoresis. The differences between receptors of cytolethal distending toxin and cholera toxin/labile toxin were exploited to develop a receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin which involved the consecutive addition of either solubilized CHO or HeLa membranes, antigen and antibody. This enzyme-linked immunosorbent assay consistently detected crude cytolethal distending toxin diluted up to 16-fold. The receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin developed in this study is a suitable alternative assay which can be performed easily in laboratories with minimal facilities and, more importantly, the results are available within a few hours as compared to times of up to 5 days in the conventional tissue culture detection of cytolethal distending toxin.

Detection of cholera toxin gene in stool specimens by polymerase chain reaction: comparison with bead enzyme-linked immunosorbent assay and culture method for laboratory diagnosis of cholera.

Stool specimens obtained from 123 hospitalized patients with acute secretory diarrhea admitted to the Infectious Diseases Hospital, Calcutta, India, were examined for isolation of Vibrio cholerae O1 by direct or enrichment plating on selective media for cholera toxin (CT) by bead enzyme-linked immunosorbent assay (bead-ELISA) and for the CT gene by polymerase chain reaction (PCR). V. cholerae O1 was isolated either by direct culture or by enrichment culture from 70 stool specimens, all of which gave positive results by PCR. Eleven specimens which were culture negative and bead-ELISA positive also gave positive results by PCR. In addition, 13 more specimens which were negative by both the culture method and bead-ELISA, were positive by PCR. With the combined results of both the culture method and the CT bead-ELISA, a confirmed laboratory diagnosis of cholera could be made from 81 stool specimens, while the combined results of the three methods, including PCR, yielded a positive result for 94 specimens examined. From these data, we conclude that PCR provides a more sensitive and specific assay for rapid diagnosis of cholera than currently available methods.

Use of a rule based graph-theoretical system in evaluating the activity of a class of nucleoside analogues against human immunodeficiency virus.

A rule based graph-theoretical system has been used to evaluate qualitatively the activity of a class of nucleoside analogues against human immunodeficiency virus (HIV). The system identifies biologically relevant vertices (atoms) in the molecular graphs of the compounds which have the biological activity of interest. The idea is to relate biological activity with the structural or substructural characteristics of the compounds from the point of view of molecular topology (connectivity). The system brings vertices of similar or close topological environment in the respective compounds together and this is reflected in the ranges of values formed by a distance based index of the vertices, the 'distance exponent index (Dx)', where x is any real number. It is found that the system makes correct prediction of the activity of all the compounds (active as well as inactives) of both training set and the test set against HIV. It is also apparent from this study that the index D-4, which has been used here, can make a useful classification of the vertices according to their molecular environment and the system can produce significant result in a small as well as diverse data base.

Virulence patterns of Vibrio cholerae non-O1 strains isolated from hospitalised patients with acute diarrhoea in Calcutta, India.

A collection of 28 strains of Vibrio cholerae non-O1 isolated during a 3-year period (1989-1991) from hospitalised patients with acute diarrhoea in Calcutta, India, were examined with regard to virulence-associated factors. Of the 28 isolates (each representing a case), 18 were isolated as the sole infecting agent; the remaining 10 were recovered as co-cultures from cases infected with V. cholerae O1. Of the strains isolated in this study, 82% could be serotyped, with serovars O5 (32.1%), O11 and O34 (14.3% each) predominant. Serovars O7, O14, O34, O39 and O97 were associated exclusively with sole infections. Two strains of V. cholerae non-O1 produced anti-cholera toxin IgG-absorbable cholera toxin (CT). Both CT-producing V. cholerae non-O1 strains hybridised with the DNA probe specific for the zonula occludens toxin (ZOT) but none of the remaining 26 strains hybridised with the ZOT probe. The majority of the strains were cytotoxic for CHO, HeLa and Vero cells, with end-point titres of 4-512. Fewer strains produced a cytotonic effect, with end-point titres of 2-16. Of the 28 strains of V. cholerae non-O1 examined, 75%, 75%, 25% and 14.3% produced haemolysin that was active against erythrocytes of rabbit, sheep (Eltor haemolysin), chicken and man, respectively. Strains that produced a haemolysin active against both rabbit and sheep erythrocytes were dominant (35.7%). Ten (35.7%) of the 28 strains examined showed cell-associated haemagglutinating activity on human blood. Of the 10 strains, nine were isolated as sole pathogen and only one strain was associated with mixed infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and sequence determination of heat-stable enterotoxin elaborated by a cholera toxin-producing strain of Vibrio cholerae O1.

Four molecular species of heat-stable enterotoxins elaborated by a cholera toxin-producing strain of Vibrio cholerae O1 were isolated from its culture supernatant. The amino acid sequence of one of the enterotoxins was determined to be Phe-Ile-Lys-Gln-Val-Asp-Glu-Asn-Gly-Asn-Leu-Ile-Asp-Cys-Cys-Glu-Ile-Cys- Cys-Asn-Pro-Ala-Cys-Phe-Gly-Cys-Leu-Asn with three intramolecular disulfide linkages. The other enterotoxins had shorter amino acid sequences in the N-terminal regions, but possessed the same sequence in their C-terminal regions including the three disulfide linkages. The enterotoxins with the shorter N-terminal sequences showed more potent toxicities, and the minimum effective dose of the longest one with 28 amino acid residues was 10-folds of that of the shortest one.

Acute secretory travellers' diarrhoea caused by Vibrio cholerae non-01 which does not produce cholera-like or heat-stable enterotoxins.

An Australian tourist suffering from severe acute watery diarrhoea and dehydration due to Vibrio cholerae non-01 was studied. The V. cholerae strain isolated from the patient belonged to serovar 05. The organism did not produce any of the conventional enterotoxins including cholera-toxin (CT) or heat-stable toxins (NAG-ST) that are known to be associated with intestinal secretion. This report suggests that toxin(s) other than CT-like or NAG-ST may be involved in the pathogenesis of diarrhoea by some V. cholerae non-01 strains.